alpha-Dystrobrevin isoforms differ in their colocalization with and stabilization of agrin-induced acetylcholine...

In skeletal muscle, alpha-dystrobrevin (alphaDB) is expressed throughout the sarcolemma with high concentrations at the neuromuscular junction. Mice lacking alphaDB display a mild muscular dystrophy and perturbations at the neuromuscular junction that include disruptions to acetylcholine receptor (AChR) cluster stability and patterning. In adult skeletal muscle, three alternatively spliced isoforms (alphaDB1, alphaDB2, alphaDB3) are expressed, while two other splice variants (alphaDB1(-) and alphaDB2(-)) are expressed only during early development. alphaDB is clearly important in AChR stabilization; however, the degree to which individual alphaDB isoforms and their specific functional domains contribute to AChR cluster stability is not fully understood. To investigate this, we established a primary muscle cell culture system from alphaDB knockout mice and stably expressed individual alphaDB isoforms using retroviral infection. A comparison between wild-type and alphaDB knockout muscle cells showed that in the absence of alphaDB, fewer AChR clusters formed in response to agrin treatment, and these AChR clusters were very unstable. Retroviral expression studies revealed that the largest isoforms (alphaDB1, alphaDB1(-), alphaDB2, alphaDB2(-)) colocalized with agrin-induced AChR clusters and rescued the AChR cluster formation defects back to wild-type levels, while only the first three isoforms fully rescued AChR cluster stability back to wild-type levels. alphaDB2(-) conferred an intermediate level of stability to the AChR clusters. In contrast, alphaDB3 showed no specific colocalization with AChR clusters and little effect on AChR cluster formation or stabilization. Twice as much syntrophin was found associated with alphaDB2 compared with alphaDB2(-) in myotubes suggesting that increased recruitment of syntrophin by alphaDB may enhance the stability of AChR clusters. Taken together, these data demonstrate that different alphaDB isoforms have different functional capabilities in the formation and maintenance of AChR clusters in muscle cells, and that these differences are likely due to the presence of different functional domains in each isoform.